The host-microbial interactions at the mucosa require a delicate, although poorly understood, balance where the presence of commensal and pathogenic bacteria requires discriminatory responses. Although the normal microbes of the gut are not pathogenic if they remain within their gut micro-niche, they will induce an inflammatory response if they cross the epithelial barrier. The persistence of translocated bacteria is thought to play an important role in the etiology and pathogenesis of inflammatory bowel disease (IBD). Inconsistencies in the literature regarding the role of bacteria-sensing genes in IBD suggest a higher level of complexity to this host-microbe interaction than has been appreciated. Some of our gaps in knowledge may be explained by the poorly defined role of one of the most abundant leukocytes in the mucosa, the B cell. B cells express Toll-like receptor (TLR) 4, the receptor for lipopolysaccharide (LPS), in IBD and likely have an important role in sensing microbes. Paradoxically, despite TLR4 expression, patient B cells generally respond poorly to E. coli LPS. In contrast, TLR4+ B cells respond to LPS molecules with characteristics of hypo-acylated lipid A, a group of ligands shown to be antagonistic for myeloid TLR4. The objective of this application is to define the potentially novel and dynamic TLR4 complex expressed by human B cells and the functional significance of B cell TLR4 responses in IBD. Our central hypothesis is that B cell TLR4 is a dynamic complex that allows recognition of hypo-acylated LPS through TRIL, a novel TLR4 co-receptor, and structural modification TLR4. Our hypothesis is based on our exciting data on disease-specific changes in the B cell TLR4 complex and our recent report demonstrating that B cells from IBD patients respond to hypo-acylated LPS by secreting IgA. We thus further hypothesize that TLR4 stimulates a restricted IgA repertoire, which exhibits broad reactivity and helps to protect the mucosa. We will test our hypothesis through two Specific Aims. In the first aim, we will define the human B cell TLR4 complex by identifying the required co-receptors through ablation and addition studies using primary B cells from patients. We will also define the effect of potential structural modification(s) of TLR4 that may affect recognition of LPS through targeted sequencing and cloning of identified B cell TLR4 variants. Data will be correlated with clinical parameters of IBD and polymorphisms in TLR4. In the second aim, we will define the mechanism(s) of TLR4-mediated IgA production by evaluating gene and kinase activation and the clonality of the B cell response through targeted sequencing of the V region of IgA. The data generated in this proposal will have direct clinical relevance by identifying critical pathways for the development of improved treatments that change the course of disease. Further, by defining how B cells recognize LPS, we will also advance the field of genome-wide association studies, human B cell immunology, and mechanism(s) of the human-microbial interface implicated in many chronic diseases. PUBLIC HEALTH RELEVANCE: Inflammatory bowel disease (IBD) is a chronic gastrointestinal disease for which we have no cure. In this proposal, we hope to identify new ways in which our immune system interacts with the friendly bacteria of the intestines in hopes of developing treatments for patients with IBD.